Methods for diagnosing infectious diseases using adsorption media

ABSTRACT

The present invention provides an in vitro method for concentrating infectious pathogens found in a biological sample obtained from an individual who is suspected of being infected with the pathogens. Provided herein is also an in vitro method for reducing or eliminating blood cells from a sample obtained from an individual suspected to being infected with an infectious pathogen. The present invention also provides a method for diagnosing malaria and a method for determining if an individual is infected with a pathogen. Provided herein is also a concentrator and a kit for use with the methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application ofPCT/US2014/064419, filed Nov. 6, 2014, which application claims priorityto U.S. Provisional Application No. 61/902,070, filed Nov. 8, 2013, thedisclosures of which are hereby incorporated by reference in theirentirety for all purposes.

BACKGROUND OF THE INVENTION

Early detection of infectious diseases is necessary to control theirspread, to direct therapy, and to improve patient outcomes. For example,early and accurate identification of outbreaks of deadly pathogens canprevent the occurrence of global pandemics. Currently many diagnosticmethods for bloodstream infections caused by, for example, viruses(including Ebola and related filoviruses) or drug-resistant bacteria,require at least 24 hours or longer to perform. There is a need in theart for a method to minimize the time needed to detect the presence of apathogen in an individual's sample. The goal is to detect the pathogenwhile it is still present at very low concentrations, if possible,before clinical symptoms are evident. Early intervention may thenminimize the intensity and duration of the infection thereby reducingmorbidity and mortality.

In some cases, the presence of cells such as blood cells (e.g., redblood cells and white blood cells) in the sample reduces the specificityand sensitivity of the assay method. At present, there exists no meansfor rapid isolation and collection of an infectious pathogen from abiological sample such that the pathogen can be identified or analyzedwhen present at very low concentration. In addition, there is a need inthe art for technologies that can improve the sensitivity of existingdiagnostic methods for detecting pathogens. The present inventionsatisfies these and other needs.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides an in vitro method forconcentrating a wide range of infectious pathogens and toxins present ina biological sample obtained from a subject who is suspected of beinginfected with said pathogens. The method comprises: (a) contacting thebiological sample obtained from the subject with a broad-spectrumadsorption media under conditions that form an adhering complexcomprising the adsorption media and said pathogens; (b) separating theadhering complex from components of the sample that are not included inthe complex while maintaining the complex, e.g., by washing the adheringcomplex with a buffer solution; and (d) collecting pathogens of theadhering complex by applying an elution buffer to the complex, therebyconcentrating the infectious pathogens in an eluent. In someembodiments, the method further comprises detecting the isolatedinfectious pathogens. In some instances, detecting the isolatedinfectious pathogens comprises a colorimetric assay, an immunoassay, anenzyme-linked immunosorbent assay (ELISA), a PCR-based assay, a pathogengrowth assay with optional staining, or a combination thereof.

In some embodiments, the wash buffer is a normal saline solution. Insome embodiments, the elution buffer is a high ionic strength orhypertonic saline solution.

In some embodiments, the biological sample is selected from the groupconsisting of whole blood, serum, plasma, urine, feces, sputum, tears,saliva, bronchial lavage fluid, other bodily fluid, and combinationsthereof.

In some embodiments, the adsorption media is a solid substrate of highsurface area having at least one polysaccharide molecular adsorbent onthe surface thereof. In some embodiments, the at least onepolysaccharide adsorbent is attached to the surface of the solidsubstrate by end-point attachment. In some embodiments, the at least onepolysaccharide adsorbent is a member selected from the group consistingof heparin, heparan sulfate, mannose, dextran sulfate, hyaluronic acid,salicylic acid, chitosan, and a combination thereof. In some instances,the mannose is D-mannose or a D-mannose polymer. In some instances, theat least one polysaccharide adsorbent is heparin and mannose.

In some embodiments, the solid substrate comprises a plurality of rigidpolymer beads. The plurality of rigid polymer beads can be rigidpolyethylene beads.

In some embodiments, the pathogens are selected from the groupconsisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale,Plasmodium malariae, Ebola virus (EBOV), non-EBOV filovirus,Flaviviridae, Streptococcus aureus, Escherichia coli,carbapenem-resistant enterobacteriaceae (CRE) bacteria, anESBL-producing pathogen, vancomycin-resistant enterococci (VRE)bacteria, Acinetobacter baumannii, Klebsiella pneumoniae, Klebsiellaoxytoca, Enterococcus faecalis, Enterococcus faecium, Candida albicans,cytomegalovirus (CMV), Adenovirus, herpes simplex virus 1 (HSV1), herpessimplex virus 2 (HSV2), and any combination thereof.

In second aspect, the present invention provides an in vitro method forreducing or eliminating blood cells from a biological sample obtainedfrom a subject who is suspected of being infected with a pathogen. Themethod comprises: (a) contacting the biological sample obtained from thesubject with an optionally broad-spectrum adsorption media underconditions to form an adhering complex comprising the adsorption mediaand a pathogen present in the sample; and (b) separating the blood cellsof the sample and the adhering complex while maintaining the adheringcomplex, thereby reducing or eliminating the blood cells from thesample. In some embodiments, step (b) further comprises washing theadhering complex with a saline solution. In some embodiments, the methodfurther comprises (c) applying an elution buffer to the adheringcomplex; and (d) collecting the pathogen of the adhering complex. Insome embodiments, the method further comprises detecting the isolatedinfectious pathogens. In some instances, detecting the isolatedinfectious pathogens comprises a colorimetric assay, an immunoassay, anenzyme-linked immunosorbent assay (ELISA), a PCR-based assay, a pathogengrowth assay, or a combination thereof.

In some embodiments, the wash buffer is a saline solution. In someembodiments, the elution buffer is a high ionic or hypertonic salinesolution

In some embodiments, the biological sample is selected from the groupconsisting of whole blood, serum, plasma, urine, feces, sputum, tears,saliva, bronchial lavage fluid, other bodily fluid, and combinationsthereof.

In some embodiments, the adsorption media is a solid substrate of highsurface area having at least one polysaccharide adsorbent on the surfacethereof. In some embodiments, the at least one polysaccharide adsorbentis attached to the surface of the solid substrate by end-pointattachment. In some embodiments, the at least one polysaccharideadsorbent is a member selected from the group consisting of heparin,heparan sulfate, mannose, dextran sulfate, hyaluronic acid, salicylicacid, chitosan, and a combination thereof. In some instances, themannose is D-mannose or a D-mannose polymer. In some instances, the atleast one polysaccharide adsorbent is heparin and mannose.

In some embodiments, the solid substrate comprises a plurality of rigidpolymer beads. The plurality of rigid polymer beads can be rigidpolyethylene beads.

In some embodiments, the pathogens are selected from the groupconsisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale,Plasmodium malariae, Ebola virus (EBOV), Streptococcus aureus,Escherichia coli, carbapenem-resistant enterobacteriaceae (CRE)bacteria, an ESBL-producing pathogen, vancomycin-resistant enterococci(VRE) bacteria, Acinetobacter baumannii, Klebsiella pneumoniae,Klebsiella oxytoca, Enterococcus faecalis, Enterococcus faecium, Candidaalbicans, cytomegalovirus (CMV), herpes simplex virus 1 (HSV1), herpessimplex virus 2 (HSV2), and any combination thereof.

In a third aspect, the present invention provides an in vitro method fordiagnosing malaria in a subject who is suspected of being infected withPlasmodium. The method comprises (a) contacting a sample obtained fromsaid subject with an adsorption media under conditions to form anadhering complex comprising the adsorption media and a cell present inthe sample which is infected with Plasmodium; (b) determining thepresence of the adhering complex by detecting a physical change to theadsorption media; and (c) predicting that the subject has malaria basedon the physical change to the adsorption media compared to a referenceadsorption media that has been contacted with a control sample. In someembodiments, the method further comprises generating a standard curve ofthe physical change to the reference media that has been contacted withthe control sample.

In some embodiments, the biological sample is selected from the groupconsisting of whole blood, serum, plasma, urine, feces, sputum, tears,saliva, bronchial lavage fluid, other bodily fluid, and combinationsthereof. In some instances, the sample is whole blood.

In some embodiments, the control sample is a sample from a healthysubject. In some embodiments, the control sample is a sample from asubject with malaria.

In some embodiments, the physical change is the color of the adsorptionmedia.

In some embodiments, the adsorption media is a solid substrate of highsurface area having at least one polysaccharide adsorbent on the surfacethereof. In some embodiments, the at least one polysaccharide adsorbentis attached to the surface of the solid substrate by end-pointattachment. In some embodiments, the at least one polysaccharideadsorbent is a member selected from the group consisting of heparin,heparan sulfate, mannose, dextran sulfate, hyaluronic acid, salicylicacid, chitosan, and a combination thereof. In some instances, themannose is D-mannose or a D-mannose polymer. In some instances, the atleast one polysaccharide adsorbent is heparin and mannose.

In some embodiments, the solid substrate comprises a plurality of rigidpolymer beads. The plurality of rigid polymer beads can be rigidpolyethylene beads.

In a third aspect, the present invention provides an in vitro method fordetermining that a subject is infected with an infectious pathogen. Themethod comprises (a) contacting a whole blood sample obtained from saidsubject with an adsorption media to form an adhering complex comprisingthe adsorption media and a pathogen present in the sample; (b)determining presence of the adhering complex by detecting a physicalchange to the adsorption media; and (c) predicting that the subject isinfected by the infectious pathogen based on the physical change to theadsorption media compared to a reference adsorption media that has beencontacted with a control sample. In some embodiments, the method furthercomprises generating a standard curve of the physical change to thereference media that has been contacted with the control sample.

In some embodiments, the biological sample is selected from the groupconsisting of whole blood, serum, plasma, urine, feces, sputum, tears,saliva, bronchial lavage fluid, other bodily fluid, and combinationsthereof. In some instances, the sample is whole blood.

In some embodiments, the control sample is a sample from a healthysubject. In some embodiments, the control sample is a sample from asubject infected with the infectious pathogen.

In some embodiments, the physical change is the color of the adsorptionmedia.

In some embodiments, the adsorption media is a solid substrate of highsurface area having at least one polysaccharide adsorbent on the surfacethereof. In some embodiments, the at least one polysaccharide adsorbentis attached to the surface of the solid substrate by end-pointattachment. In some embodiments, the at least one polysaccharideadsorbent is a member selected from the group consisting of heparin,heparan sulfate, mannose, dextran sulfate, hyaluronic acid, salicylicacid, chitosan, and a combination thereof. In some instances, themannose is D-mannose or a D-mannose polymer. In some instances, the atleast one polysaccharide adsorbent is heparin and mannose.

In some embodiments, the solid substrate comprises a plurality of rigidpolymer beads. The plurality of rigid polymer beads can be rigidpolyethylene beads.

In some embodiments, the pathogens are selected from the groupconsisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale,Plasmodium malariae, Ebola virus (EBOV), Streptococcus aureus,Escherichia coli, carbapenem-resistant enterobacteriaceae (CRE)bacteria, an ESBL-producing pathogen, vancomycin-resistant enterococci(VRE) bacteria, Acinetobacter baumannii, Klebsiella pneumoniae,Klebsiella oxytoca, Enterococcus faecalis, Enterococcus faecium, Candidaalbicans, cytomegalovirus (CMV), herpes simplex virus 1 (HSV 1), herpessimplex virus 2 (HSV2), and any combination thereof.

In another aspect, the present invention provides an in vitro method fordetecting an infectious pathogen present in a biological sample obtainedfrom a subject who is suspected of being infected, the methodcomprising: (a) contacting the biological sample obtained from thesubject with an adsorption media under conditions to form an adheringcomplex comprising the adsorption media and said pathogen; (b)separating the adhering complex from components of the biological samplethat are not included in the complex while maintaining the complex; and(c) collecting the pathogen of the adhering complex, wherein thepathogen or a component of the pathogen is collected after using anenzymatic digestion while the pathogen is resident on the adsorptionmedia.

In some embodiments, the digestion is with one or more enzymes such as aprotease or DNase while the pathogen (e.g. virus) is still adsorbed tothe media.

These and other aspects, objects and embodiments will become moreapparent when read with the detailed description which follows.

BRIEF DESCRIPTION OF THE DRAWING

The FIGURE shows an embodiment of a concentrator of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for infectious disease detectionand diagnosis. Advantageously, the present invention can be used toisolate bacteria, viruses, cytokines and other pathogens (e.g.,parasites) from a patient sample which can be used in the earlydetection of an infection. In another aspect of the present invention,cells and other non-analytes that can interfere with analyte detectionassay can be removed from the sample using the adsorption mediadescribed herein.

Also provided herein are simple chromogenic assay methods for detectingcirculating pRBCs and Plasmodium from a sample taken from a humansubject. The method also can be used to monitor malaria progressionduring the initiation and/or the completion of the anti-malaria therapy.

It has been surprisingly discovered that parasitemia in pRBCs can bedetected by contacting the infected blood with a polysaccharide (e.g.,heparin, or heparan sulfate) that has been covalently bound to anadsorption media. The parasites and pRBCs become bound onto theadsorption media, and in turn alter the color of the media. Thus, avisible color change indicates that the subject has a malaria infection.

I. DEFINITIONS

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The term “malaria infection” refers to an infection caused by theparasitic protozoans of the genus Plasmodium, such as, but not limitedto, P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi.

The term “adsorption media” refers to a material to which a cell,polypeptide, polynucleotide, chemical molecule, biological molecule canadhere to the surface thereof.

The term “adhering complex” refers to a complex of at least twomolecules wherein the first molecule is attached (e.g., linked, coupledor bound) to the surface of a substrate and the second molecule isattached to the first molecule.

The term “color change” refers to a change from a first color to asecond color. If there is no detectable difference in the first colorand the second color, then a color change is not indicated.

The term “visible spectrum” refers to the portion of the electromagneticspectrum, e.g., about 390 to 7000 nm that can be detected by the humaneye. Light in the near infrared, medium wavelength infrared, and farwavelength spectrums are not visible by the human eye.

The term “healthy control” refers to an individual who does not have aninfection. The term “positive control” refers to an individual with aninfection caused by the pathogen of interest.

The term “high surface area” refers to the property of having a largespecific surface area to volume ratio.

The term “adsorbent” refers to a solid substrate with a chemicalcompound, a biological molecule, or a material that is attached (e.g.,linked, coupled or bound) thereto. In certain instances, the adsorbentis the solid substrate itself. In one embodiment, an adsorbent is apolymer resin with a polysaccharide bound thereto.

The term “rigid polymer bead” refers to a bead, granule, pellet, sphere,particle, microcapsule, sphere, microsphere, nanosphere, microbead,nanobead, microparticle, nanoparticle, and the like that is made from apolymer resin.

The term “carbohydrate” refers to a molecule containing carbon, hydrogenand oxygen atoms, and usually with the empirical formula C_(x)(H₂O)_(y),where x and y are different numbers. Examples of carbohydrates includesmonosaccharides, disaccharides, oligosaccharides, and polysaccharides.

The term “polysaccharide” refers to a molecule comprising manymonosaccharide units joined together by glycosidic bonds, and having anempirical formula of C_(x)(H₂O)_(y), where x is between 200 to about3000.

The term “anti-malaria therapy” refers to a treatment such as apharmaceutically effective agent intended to relieve or remedy amalarial infection.

II. DETAILED DESCRIPTION OF EMBODIMENTS A. Adsorption Media

The adsorption media of the present invention provides a surface toattach a polysaccharide adsorbent that can bind to analytes/pathogensand pRBCs. In some embodiments, the adsorption media includes a solidsubstrate with a high surface area having at least one polysaccharideadsorbent on the surface thereof. The solid substrate can be made of,for example, but not limited to, polyethylene, polystyrene,polypropylene, polysulfone, polyacrylonitrile, polycarbonate,polyurethane, silica, latex, glass, cellulose, cellulose acetate,crosslinked dextran, crosslinked agarose, chitin, chitosan, crosslinkeddextran, crosslinked alginate, silicone, Teflon®, fluoropolymer, andother synthetic polymers. The solid substrate with a high surface areacan be a plurality of adsorbent monolayers, filters, membranes, solidfibers, hollow fibers, particles, or beads. Optionally, the solidsubstrate can be present in other forms or shapes or configured articlesproviding a large surface area, sufficient to bind a detectable quantityof analyte. The substrate can be configured into different shapes.

Useful substrates for creating the media include, but are not limitedto, non-porous rigid beads, particles, or packing, reticulated foams, arigid monolithic bed (e.g. formed from sintered beads or particles), acolumn packed with woven or nonwoven fabric, a column packed with a yarnor solid or hollow dense (not microporous) monofilament fibers, a flatfilm or barrier membrane, a spiral wound cartridge formed from flat filmor dense membrane, or a combination of media such as a mixed bead/fabriccartridge.

In certain instances, a suitable substrate is one that is initiallymicroporous, but becomes essentially nonporous when the surface istreated before, during or after the creation of adsorption sites, e.g.,via end-point-attached one or more polysaccharide adsorbents. In oneembodiment, the substrate is in the form of solid beads or particles.

In certain instances, the solid substrate is a plurality of rigidpolymer beads such as polyethylene, polystyrene, polypropylene,polysulfone, polyacrylonitrile, polycarbonate, polyurethane, silica,latex, glass, cellulose, crosslinked agarose, chitin, chitosan,crosslinked dextran, crosslinked alginate, silicone, fluoropolymer, andsynthetic polymer beads. Preferably, the rigid polymer beads arepolyethylene beads.

In certain aspects, the adsorbtion media (e.g. beads) can be affixed orshaped into a dip stick, a strip, a polymer stick, magnetic beads, orother forms. In fact, the substrate can be configured into differentshapes and sizes. The adsorption media can be affixed to or configuredinto a dip stick or dip strip. For example, a polymer dip stick issuitable for use in the present invention such as one made of rough orsintered ultra high molecular weigh polyethylene (UHMWPE) particles, orother polymers that do not leach or interfere with detection assays.Other substrates or surfaces include metallic, cellulosic, e.g. paper,open-cell foam, magnetic, fibers, and the like, wherein the adsorptionmedia is affixed or coated to these materials. As long as the surfacematerial or substrate for the adsorption media does not releaseinterfering substances, it is suitable for use in the present invention.High-surface areas are preferred for maximizing capture of the analyte.

The size of the solid substrate can be selected according to the volumeof the test sample used in the assay or other parameters. In someembodiments, the plurality of rigid polymer beads has an average outerdiameter of about 1 μm to about 1 mm, e.g., 1 μm, 2 μm, 3 μm, 4 μm, 5μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 45μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, and1 mm. In other embodiments, the plurality of rigid polymer beads has anaverage diameter of about 10 μm to about 200 μm, e.g., 10 μm, 15 μm, 20μm, 25 μm, 30 μm, 35 μm, 45 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80μm, 85 μm, 90 μm, 95 μm, 100 μm, 105 μm, 110 μm, 115 μm, 120 μm, 125 μm,130 μm, 135 μm, 140 μm, 145 μm, 150 μm, 155 μm, 160 μm, 165 μm, 170 μm,175 μm, 180 μm, 185 μm, 190 μm 195 μm, and 200 μm. Useful beads have asize ranging from about 100 to above 500 microns in diameter such as100, 200, 300, 400, or 500 microns. The average size of the beads can befrom 150 to 450 microns. See, for example, WO 2011/068897, the entirecontents of which are hereby incorporated by reference.

The surface of the solid substrate can be functionalized to allow thecovalent attachment of the polysaccharide adsorbent described herein. Insome embodiments, the surface of the solid substrate has at least onechemical group, such as an amine group.

The adsorption media can be contained within a housing, such as asyringe, a column, cartridge, tube, centrifuge tube, and the like, orany vessel. In some embodiments, the vessel is a particular shape andsize such that RBCs that are not captured onto polysaccharide boundadsorption media can be removed without disturbing the parasitized RBCsattached to the media.

The housing comprising the adsorption media can contain more than onetype of adsorption media. In some embodiments, the different media islayered in a parfait-type arrangement within the housing such that thesample, e.g., whole blood, contacts the different media in series orparallel flow. One arrangement of the different media within a cartridgeis to position a first adsorption media at the entrance and/or the exitof the cartridge, with an optionally blended region containing thesecond adsorption media interposed between the entrance and exitregions. In the case of media in fiber form, a mixed woven, knitted, ornon-woven structure can be prepared by methods well known in the textileindustry to form fabric from the mixed fiber. Alternatively, a yarn canbe prepared from finer multifilament yarn or monofilament made from twoor more fibers with different surface chemistries, as long as one fibertype contains a surface that actively prevents blood clotting oncontact. The mixed-fiber yarn can then be used to prepare fabric forblood contact.

B. Polysaccharide Adsorbents

In some embodiments, the polysaccharide adsorbent is heparin, heparansulfate, mannose, dextran sulfate, hyaluronic acid, sialic acid,chitosan, and a combination thereof. In some instances, one or moredifferent polysaccharide adsorbents, e.g., 1, 2, 3, 4, 5 or moredifferent polysaccharide adsorbents, are attached to the solid substrateof the adsorption media. In some embodiments, the absorbent is heparin.In some embodiments, the absorbent is heparan sulfate. In otherembodiments, the absorbent is mannose. In another embodiment, theabsorbent is dextran sulfate. In some instances, the polysaccharideadsorbents are heparin and mannose. In some instances, thepolysaccharide adsorbents are heparan sulfate and mannose. In otherinstances, the polysaccharide adsorbents are heparin and dextransulfate. In yet other instances, the polysaccharide adsorbents aremannose and dextran sulfate.

In some embodiments, more than 1 adsorbent, e.g., 2 absorbents, areattached onto a single solid substrate. In some instances, the ratio ofthe two adsorbents (A and B) are in the range of 1:99 to 99:1. In otherembodiments, the substrate is coated with about 1-50% of adsorbent A andabout 1-50% of adsorbent B.

In some embodiments, mannose used as an adsorbent is a reducing sugar oris a non-reducing sugar (e.g., a mannoside). Suitable mannoses include,but are not limited to, D-mannose, L-mannose,p-aminophenyl-α-D-mannopyranoside, a mannose containing polysaccharide,and mannan. The term “mannose” also includes a polymer of mannose suchas mannan. Mannan refers to a plant polysaccharide that is a linearpolymer of the sugar mannose. Plant mannans have β(1-4) linkages. Mannancan also refer to a cell wall polysaccharide found in yeasts. This typeof mannan has a α(1-6) linked backbone and α(1-2) and α(1-3) linkedbranches.

In one embodiment, the mannose is bound by end-point attachment to thesolid substrate. In another embodiment, the mannose is attached to thesubstrate by multi-point attachment.

In other instances, mannose is a polymer of mannose such as mannan.Mannan refers to a plant polysaccharide that is a linear polymer of thesugar mannose. Plant mannans have β(1-4) linkages. Mannan can also referto a cell wall polysaccharide found in yeasts. This type of mannan has aα(1-6) linked backbone and α(1-2) and α(1-3) linked branches.

Red blood cells infected by P. falciparum express erythrocyte membraneprotein 1 (PfEMP1) which can bind to specific binding molecules presenton the surface of endothelial cells and other RBCs. The method providedherein is based in part on the ability of parasitized red blood cells tobind select binding molecules, such as polysaccharides. Moreover, whenthese binding molecules are bound to the surface of an adsorption media,the media can be used to separate pRBCs from a patient sample, which inturn alters the color of the adsorption media. Thus, the presence of amalaria infection can be determined by contacting a patient sample witha binding molecule bound to an adsorption media.

Molecules that can bind pRBCs and in particular PfEMP1 include, but arenot limited to, polysaccharides, such as glycoaminoglycans, e.g.,heparin, heparan sulfate, and chondroitin sulfate A (CSA), sialic acid,the complement receptor 1 (CR1), the ABO blood group antigens A and B,ICAM-1, CD36, trombospondin (TSP), endothelial protein C receptor(EPCR), E-selectin, vascular cell adhesion molecule 1 (VCAM-1), plateletendothelial cell adhesion molecule 1 (PECAM-1), endothelial leukocyteadhesion molecule 1 (ELAM-1), serum proteins IgG/IgM, and fibrinogen,carbohydrates with mannose groups, lectins, and chitosans. AdditionalpRBC binding molecules include hyaluronate, peptidoglycans,glycoproteins, glycolipids, glycans, glycosylphosphatidylinositol (GPI)glycans, and hyaluronic acid, and other neuraminic acids.

The binding molecules provided above can be used to adsorb pRBCs onto asurface. In some embodiments, at least one polysaccharide adsorbent isattached to a solid substrate of high surface area to form an adsorptionmedia. In some embodiments, the polysaccharide adsorbent is heparin,heparan sulfate, hyaluronic acid, sialic acid, carbohydrates withmannose sequences, and chitosan. In one embodiment, the polysaccharideadsorbent is heparin.

In addition to mixed carbohydrates, it is possible to include additionalbinding moieties specific for the analyte. These include proteins,peptides, antibodies, affibodies, nucleic acids and other specificbinding moieties (See, US Pat. Pub. 2003/0044769, incorporated byreference herein).

C. Attachment of Polysaccharide Adsorbents onto the Surface of theAdsorption Media

Polysaccharides can be linked onto the surface of the adsorption mediaby single covalent bond end-point attachment (e.g., covalent attachmentthrough the terminal residue of the heparin molecule). A single covalentattachment at the terminal group of the molecule to be attached, ascompared to non-covalent attachment or multi-point attachment,advantageously provides better control of the orientation of theimmobilized molecules while maximizing their surface density. Inparticular, the end-point attachment of these long chain carbohydratesprovides a brush-type molecular surface architecture that leads to ahigher concentration of accessible positions on the carbohydrateoligomers available for analyte/pathogen binding. In some instances,pRBCs attach to full-length heparin (e.g., heparin with a mean molecularweight of more than 10 kDa) coated surfaces much more efficiently thanto conventional surfaces coated with heparin fragments, as is generallyemployed in the prior art.

Covalent attachment of a carbohydrate (e.g., a polysaccharide) to asolid substrate provides control of parameters such as surface densityand orientation of the immobilized molecules as compared to non-covalentattachment. These parameters have been shown to provide adsorbatebinding to the immobilized carbohydrate molecules. In certainembodiments, the surface concentration of the carbohydrate on the solidsubstrate is in the range of 0.01 to about 0.5 μg/cm², such as 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13,0.14, 0.15, 0.16, 0.17, 0.18, 0.19 or 0.2 μg/cm². In other embodiments,the surface concentration of the absorbent(s) on the solid substrate isin the range of 0.001-2.0 μg/cm². In another embodiment, the surfaceconcentration of the absorbent(s) on the solid substrate is in the rangeof 0.005-0.5 μg/cm².

In some embodiments, the surface concentration of the adsorbent on thesolid substrate is in the range of 1 μg/cm² to 20 μg/cm², e.g., 1μg/cm², 2 μg/cm², 3 μg/cm², 4 μg/cm², 5 μg/cm², 6 μg/cm², 7 μg/cm², 8μg/cm², 9 μg/cm², 10 μg/cm², 11 μg/cm², 12 μg/cm², 13 μg/cm², 14 μg/cm²,15 μg/cm², 16 μg/cm², 17 μg/cm², 18 μg/cm², 19 μg/cm², and 20 μg/cm². Inother embodiments, the surface concentration of the adsorbent on thesolid substrate is in the range of 5 μg/cm² to 15 μg/cm², e.g., 5μg/cm², 6 μg/cm², 7 μg/cm², 8 μg/cm², 9 μg/cm², 10 μg/cm², 11 μg/cm², 12μg/cm², 13 μg/cm², 14 μg/cm², and 15 μg/cm².

In some embodiments, mannose, derivatives of mannose and oligomers ofmannose are reductively coupled to primary amines on aminated substratessuch as aminated beads by reductive amination. Coupling of the openaldehyde form of a reducing mannose to a bead results in a stablesecondary amine. Non-reducing mannoses having a reactive amine can becoupled to a bead with an intermediate having an aldehyde functionality.For instance, mannose is attached to an amine containing substrate by(a) contacting an aminated substrate with an aqueous solution containinga mannose to form a Schiff base intermediate; and (b) contacting theSchiff base with a reducing agent to attach the mannose. In someembodiments, if the mannose is a nonreducing mannose, an intermediatealdehyde (e.g., glutardialdehyde) is attached to the amine substrateprior to the non-reducing mannose.

Mannose can be dissolved in aqueous solution such as an acidic aqueoussolution. The mannose aqueous solution is contacted with an aminatedsubstrate such as an aminated bead. A Schiff's base is generated. TheSchiff's base is thereafter reduced with a reducing agent. The reducingagent can be, for example, sodium cyanoborohydride or sodiumborohydride. In certain instances, the solid substrate is also reactedwith heparin having a reactive aldehyde functionality.

For heparin attachment, a more reactive aldehyde function in thereducing terminal residue can be achieved by partial, nitrous aciddegradation. This shortens the reaction time, but the immobilizedheparin will have a lower molecular weight. The coupling is performed inaqueous solution, by reductive amination (cyanoborohydride).

Covalent attachment of full-length heparin molecules to a surface can beachieved by the reaction of an aldehyde group of the heparin moleculewith a primary amino group present on the surface of the adsorptionmedia. An inherent property of all carbohydrates is that they have ahemiacetal in their reducing end. This acetal is in equilibrium with thealdehyde form and can form Schiff's bases with primary amines. TheseSchiff's bases may then be reduced to stable secondary amines. In someembodiments, full-length heparin is surface immobilized onto the solidsubstrate by covalent conjugation. In other embodiments, full-lengthheparin is covalently attached to said adsorption media via a stablesecondary amino group.

In some embodiments, the immobilized full-length heparin molecules havea mean molecular weight of more than 10 kDa. In other embodiments, theimmobilized heparin molecules have a mean molecular weight of more than15 kDa. In another embodiment, the immobilized heparin molecules have amean molecular weight of more than 21 kDa. In yet another embodiment,the immobilized heparin molecules have a mean molecular weight of morethan 30 kDa. Preferably, the immobilized heparin molecules have a meanmolecular weight within the range of 15-25 kDa. The mean molecularweight may also be higher, such as in the range of 25-35 kDa.

In certain instances, various methods of making adsorbents and theadsorbents per se are disclosed in U.S. Pat. Nos. 8,663,148 and8,758,286; and U.S. Application Publication Nos. 2009/0136586,2012/0305482, and US 2014/231357, the disclosures of which are hereinincorporated by reference for all purposes.

D. Analytes/Pathogens that Bind Adsorbents Bound to the Adsorption Media

The adsorbents attached to the adsorption media can be used to bind to aanalyte/pathogen of interest in a sample. In some embodiments, thesample is selected from the group consisting of whole blood, serum,plasma, urine, feces, sputum, tears, saliva, bronchial lavage fluid,other bodily fluid, and combinations thereof. In some instances, thesample is whole blood from a subject, e.g., a human subject.

The analyte/pathogen can include, but is not limited to, Plasmodiumfalciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae,Ebola virus (EBOV), Streptococcus aureus, Streptococcus pyogenes,Streptococcus pneumonia, Neisseria meningitides, Pseudomonas aeruginosa,Escherichia coli, carbapenem-resistant enterobacteriaceae (CRE)bacteria, (e.g., carbapenem-resistant Escherichia coli andcarbapenem-resistant Klebsiella pneumoniae), an ESBL-producing pathogen(e.g., ESBL-producing E. coli, ESBL-producing K. pneumonia, andESBL-producing K. oxytoca), vancomycin-resistant enterococci (VRE)bacteria (e.g., vancomycin-resistant E. faecalis andvancomycin-resistant E. faecium), Acinetobacter baumannii, Klebsiellapneumoniae, Klebsiella oxytoca, Enterococcus faecalis, Enterococcusfaecium, Candida albicans, cytomegalovirus (CMV), herpes simplex virus 1(HSV1), herpes simplex virus 2 (HSV2) and any combination thereof.

In certain other instances, the analyte/pathogen of interest includes,but is not limited to, Hepatitis A Virus (HAV), Hepatitis B Virus (HBV),Hepatitis C Virus (HCV), Hepatitis D Virus (HDV), Hepatitis G Virus/GB-CVirus (HGV/GBV-C), Human Immunodeficiency Virus types 1 and 2 (HIV-1/2),Human T-cell Lymphotropic Virus types I and II (HTLV-I/II),Cytomegalovirus (CMV), Epstein-Barr Virus (EBV), TT Virus (TTV), HumanHerpes virus type 6 (HHV-6), SEN Virus (SEN-V), and Human Parvovirus(HPV-B19).

Additional viruses of interest include, but are not limited to, humanherpes virus type 7 (HHV-7), human herpes virus type 8 (HHV-8),influenza type A viruses, including subtypes H1N1 and H5N1, severe acuterespiratory syndrome (SARS) coronavirus, and RNA viruses that causeshemorrhagic fever, such as Arenaviridae (e.g., Lassa fever virus (LFV)),Filoviridae (e.g., Ebola virus (EBOV) and Marburg virus (MBGV));Bunyaviridae (e.g., Rift Valley fever virus (RVFV) and Crimean-Congohemorrhagic fever virus (CCHFV)); and Flaviviridae (West Nile virus(WNV), Dengue fever virus (DENY), yellow fever virus (YFV), and (GBvirus C (GBV-C), formerly known as Hepatitis G virus (HGV)).

In certain instances, the bacteria include, but are not limited to,Treponema Pallidum (TP, the agent of syphilis), Yersinia Enterocolitica,and Staphylococcus and Streptococcus species (common agents of bacterialcontamination), and parasites such as Plasmodium species (the agent ofmalaria), Trypanosoma Cruzi (agent of Chagas' disease), and BabesiaMicroti (agent of babesiosis). Additional bacteria include, but are notlimited to, Staphylococcus epidermidis, Bacillus cereus, Eikenellacorroders, Listeria monocytogenes, Streptococcus agalactiae, Haemophilusinfluenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Bacteroidesfragilis, Bacillus anthracis, Yersinia pestis, Yersinia enterocolitica,Francisella tularensis, Brucella abortus, Serratia marcescens, Serratialiquelaciens, Pseudomonas fluorescens and Deinococcus radiodurans.

In addition, emerging blood-borne pathogens such as Candida sp.,including Candida albicans, Aspergillus sp., including Aspergillusfumigatus, Hepatitis E Virus (HEV), Human Herpes virus type 8 (HHV-8),Borrelia Burgdorferi (agent of Lyme disease), and the unknown agent ofCreutzfeldt-Jakob disease (CJD) can be detected.

Pathogens known to bind to heparin/heparan sulfate can used in themethods described herein. Non-limiting examples of such pathogensinclude bacteria, e.g., Bacillus anthracis, Bacillus cereus, Borreliaburgdorferi, Bordetella pertussis, Chlamydia pneumoniae, Chlamydiatrachomatis, Haemophilus influenzae nontypable, Helicobacter pylori,Listeria monocytogenes, Mycobacterium tuberculosis, Neisseriagonorrhoaea, Neisseria meningitidis, Orientia tsutsugamushi,Porphyromonas gingivalis, Pseudomonas aeruginosa, Staphylococcus aureus,Streptcoccus agalactiae, Streptococcus pyogenes, Steptococcuspneumoniae, Yersinia enterocolitica; viruses, e.g., adeno-associatedvirus type 2, adenovirus, coronavirus, coxsackievirus, cytomegalovirus,Dengue virus, FMDV, HSV1, HSV2, hepatitis B viris, hepatitis C virus,HHV8, HIV1, HPV, HTLV1, Japanese encephalitis virus, pseudorabies virus,respiratory syncytial virus, rhinovirus, sindbis virus, vaccinia virus,West Nile virus, Yellow Fever virus; parasites, e.g., Giardia lambia,Leishmania spp., Encephalitozoon spp., Neospora caninum, Plasmodiumspp., Toxoplasma gondii, Trypanosona cruzi, and prions. See, e.g.,Barlett and Park, “Chapter 2 Heparan Sulfate Proteoglycans in Infection”in M. S. G. Pavao, ed. Glycans in Diseases and Therapeutics, Biology ofExtracellular Matrix, Heidelberg:Spring-Verlag, 2011.

E. Methods for Detecting Analytes/Pathogens

Provided herein is a method for reducing blood cells in a biologicalsample from an individual who is infected or is suspected of beinginfected with said analyte/pathogen. The method can be used to removeblood cells which can interfere with the sensitivity of conventionaltechniques used to detected the analyte/pathogen of interest. The methodcan include exposing the sample that includes blood cells to theadsorption media described herein under conditions to form an adheringcomplex that includes the analyte/pathogen and the adsorption media. Theblood cells of the sample can be separated from the adhering complex by,for example, gravity, or other means that maintain the adhering complex.In some instances, a saline solution, such as a normal saline solution,e.g., a solution of about 0.90% w/v of NaCl, about 300 mOsm/L, a 0.01Nsaline solution, or a similar solution, is applied to the adheringcomplex including adsorption media to wash the remaining blood cellsaway.

Also provided herein is a method for concentrating analytes/infectiouspathogens in a sample obtained from an individual who is infected or issuspected of being infected with said analytes/pathogens. The sample canbe exposed to the adsorption media described herein under conditions toform an adhering complex that includes the adsorption media and theanalytes/pathogens. Components of the sample that are not part of theadhering complex can be separated by, for example, gravity, withoutdisturbing the adhering complex. The adhering complex including theadsorption media can be washed with a wash buffer such as a normalsaline solution or a solution that maintains (preserves) the complex. Insome instances, a normal saline solution is a solution of about 0.90%w/v of NaCl, about 300 mOsm/L, a 0.01N saline solution, or a similarsolution. The complex can be disrupted to separate theanalytes/pathogens from the adsorption media by applying an elutionbuffer, such as a high ionic saline solution, to the complex. In someinstances, a high ionic saline solution is a 2N saline solution. Inother cases, the elution buffer is a buffer that can disrupt the bindingof the polysaccharide adsorbent and the analyte/pathogen. In someembodiments, different analytes/pathogens can be eluted from theadsorption media in different fractions by using various elution buffersselected for the particular analyte/pathogen of interest. Similar to achromatography column, specific analytes/pathogens can be eluted atdifferent time in different pools. The analyte/pathogen of interest canbe eluted in one or more fractions.

This process concentrates analytes/pathogens, which can then beidentified and analyzed using standard methods known to those in theart. For example, bacterial, viral, fungal, protozoan, parasitic, andmicrobial pathogens can be detected using assays, such as, colorimetricassays, immunoassays (e.g., sandwich assays or dipstick assay),enzyme-linked immunosorbent assays (ELISAs), PCR-based assays (e.g.,RT-PCR, qPCR, TagMan® assays), pathogen growth assays (e.g., drugresistance or antibiotic resistance assays), and variants thereof. Forinstances, HCV RNA is detected using RT-PCR using a standard kit, suchas COBAS® AmpliPrep/COBAS® TagMan® HCV test (Roche Diagnostics,Indianapolis, Ind.) and RealTime HCV Genotype II (Abbott Molecular,Abbott Park, Ill.). Ebola virus can be detected using, for example,BioFire Diagnostics' FilmArray and the CDC's Ebola Virus VP40 Real-timeRT-PCR Assay.

Drug-resistant pathogens can be detected by culturing the pathogen inthe presence of the drug. For example, a pathogen suspected to be acarbapenem-resistant Klebsiella or E. coli can be used to inoculate agrowth media containing ertapenem or meropenem. After properingculturing, the incubated broth culture can be subculture onto aMacConkey agar plate. The following day, the agar plate can be examinedfor lactose-fermenting (pink-red) colonies. In addition, the isolatedcolonies can be screened using a phenotypic test for carbapenemaseproduction, such as the Modified Hodge Test (MHT).

In certain instances, the analyte of interest can be detected and/oridentified using typical laboratory techniques such as enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and affinitychromatography. For example, in certain instances a commercial availableELISA kit can be used, or is easily developed. In the ELISA, a specificantibody is passively absorbed to a plate. The nonspecific sites areblocked with a protein solution which has no active part in the specificimmunochemical reaction of a particular assay. A specificanalyte/pathogen is captured by the antibody on the surface and thendetected by another antibody with an enzyme label. The enzyme label isreacted with specific reagents and the presence of the analyte isdetected and identified.

In certain other instances, a quartz crystal microbalance (QCM) can beused to detect the analyte adsorbed to the surface. A quartz crystalmicrobalance is a very sensitive and inexpensive detector/sensor thatproduces a frequency shift in quartz crystal vibration when moleculesinteract with its surface. The QCM detects a physical change such as amass per unit area by measuring the change in frequency of a quartzcrystal resonator. In liquid, the QCM is highly effective at determiningthe affinity of molecules to surfaces functionalized with recognitionsites. Larger entities such as viruses and microbes can also berecognized. For example, QCMs can be coupled to antibodies, allowing forthe selective sensing of an antibody conjugate. In certain instances,the eluent contains an analyte or pathogen of interest. The QCM hasconjugated thereto an antibody specific for the analyte. By contactingthe QCM with eluent containing the analyte/pathogen, the analyte bindsto the surface of the QCM, and the QCM senses the binding, presence andits identity. Other affinity molecules can also be bound to the surfacesuch as nucleic acids, carbohydrates, peptides, proteins and the like.

In still other instances, surface plasmon resonance (SPR) techniques canbe used to detect the presence and or identity of the analyte/pathogen.Surface plasmon is a quantum name for an electric charge density wavethat propagates on an interface between a metal and a dielectric, justlike photon is a quantum name for a light wave. Surface plasmonsresonate upon excitation by electromagnetic radiation entering aninterface of metallic material and a dielectric material. The surfaceplasmon responds to changes in the environment in close proximity to theinterface. This fact makes surface plasmon resonance useful for thedetection of biomolecular interactions. Like QCM, it is possible tolabel the SPR surface with either an antibody or binding moiety such asan antigen of an analyte/pathogen of interest.

The method for concentrating analytes/pathogens from blood, e.g., wholeblood or serum, or for reducing blood cells from blood can includecontacting the blood with a solid, essentially non micro-poroussubstrate which has been surface treated with a polysaccharide adsorbentthat has a binding affinity for the analytes/pathogens to be removed(the adsorbates). The size of the interstitial channels within saidmedia is balanced with the amount of media surface area and the surfaceconcentration of binding sites on the media in order to provide adequateadsorptive capacity while also allowing relatively high flow rates ofblood through the adsorbent media. The result is that transport ofadsorbates to the binding sites on the media occurs largely by forcedconvection, not by slow diffusion. By (forced) convection is meant, forexample, flow produced by a pressure gradient generated by a pump, or asyringe by the application of external pressure to a flexible container(or internal pressure to a rigid container), by a gravity head/elevationdifference, or by the difference in arterial pressure and venouspressure in the patient being treated with an extracorporeal device.

F. Method of Diagnosing a Malaria Infection

Provided herein are methods of using an adsorption media comprising aheparin-immobilized solid substrate to determine the presence of amalaria infection. A sample taken from an individual suspected of havingmalaria can be tested for infected red blood cells (RBCs) or parasitizedRBCs using the method described herein. This method is based on theobservation that heparan sulfate binding proteins located on the cellsurface of parasitized RBCs can adhere to heparin, heparan sulfate orother polysaccharides that are bound on a solid surface. In addition,the method can be utilized to determine whether the individual should beadministered an anti-malaria therapy. Furthermore, the method can beused to monitor the progression of malaria in an individual receivingsaid therapy.

A sample can be obtained from a subject (e.g., human individual)suspected of having a malaria infection. In some embodiments, the sampleis a member selected from the group consisting of whole blood, serum,plasma, urine, sputum, bronchial lavage fluid, tears, nipple aspirate,lymph, saliva, cerebral spinal fluid, tissue and combinations thereof.Preferably, the sample is whole blood.

The sample is then contacted with the adsorption media under conditionsto allow the polysaccharide adsorbent on the surface of the solidsubstrate to bind to a parasitized red blood cell if present in thesample, to form an adhering complex.

In some instances, after the formation of the adhering complex thesample and the adsorption media are separated without disrupting orseparating the complex from the media.

The presence of the adhering complex is detected by inspecting the colorof the adsorption media within the visible light spectrum. (e.g.,wavelength of about 400 nm to about 700 nm). In some instances, thedetectable color is within the wavelength range of about 625 nm to 740nm, or range corresponding to the color red or variations thereof. Thecolor of the adsorption media can be determined by using an opticaldetector of visible light or the human eye.

In certain instances, it is possible to concentrate the analyte (e.g.,virus, pathogen, bacteria, cytokine or adhering complex) on the mediaand detect the analyte by a color change of the media. In an alternativeembodiment, it is possible to concentrate the analyte to be detected byeluting the analyte from the column, concentrating the analyte andmeasuring a color change of media. Other forms of detection are alsopossible.

In some embodiments, the color of the test adsorption media is comparedto a control adsorption media. In some embodiments, the control media(e.g., negative control for infection) is an adsorption media that hasbeen exposed to a sample taken from a healthy control such as a subjectwho does not have a malaria infection. In other embodiments, the controlmedia (e.g., positive control) is an adsorption media that has beenexposed to a sample taken from a subject having a malaria infection(e.g., uncomplicated or severe malaria infection).

If the adsorption media from the test subject has a similar color tothat of the healthy control, then the assay indicates the absence of theadhering complex. Yet, if the adsorption media from the test subject hasa similar color to that of the positive control, then the assayindicates the presence of the adhering complex, and thus a malariainfection.

If the presence of the adhering complex is detected, then the subjectcan be administered an anti-malaria therapy. Non-limiting examples of ananti-malaria therapy include chloroquine (Aralen), quinine sulfate(Qualaquin), hydroxychloroquine (Plaquenil), atovaquone-proguanil(Malarone), artemether-lumefantrine (Coartem), mefloquine (Lariam),primaquine, amodiaquine, quinine, quinidine, doxycycline, clindamycin,sulfonamides, such as sulfadoxine and sulfamethoxypyridazine,pyrimethamine, halofantrine, artemisinin, artemisinin derivativesthereof, and combinations thereof. Artemisinin derivatives includeartemether, artesunate, dihydroartemisinin, artenimol, artemotil, andarteether. Other artemisinin derivatives that are suitable anti-malariadrugs are found in, for example, U.S. Pat. Nos. 8,722,910; 8,481,757;8,304,440; 7,851,512; 7,776,911; 7,084,132; 6,586,464; 6,362,219; and6,306,896; and U.S. Patent Appl. Pub. Nos. 2012/0258945, 2013/0072513,2013/071474, 2014/011829, 2014/011830, and 2014/256761.

G. Infected RBCs can Attach to RBC Binding Molecules on the Surface ofthe Adsorption Media

Infected red blood cells can be retained on the surface of an absorptionmedia bound by a polysaccharide such as but not limited to, heparansulfate, heparin, condroitin sulfate, and derivatives thereof.Non-limiting examples of infecting agents, such as viruses and pathogens(e.g., bacteria and parasites) that can bind to heparan sulfate, heparinand analogs thereof include bacteria, e.g., Bacillus anthracis, Bacilluscereus, Borrelia burdorferi, Bordetella pertusis, Chlamydia pneumoniae,Chlamydia trachomatis, Hemophilus influenzae, Helicobacter pylori,Listeria monocytogenes, Mycobacterium tuberculosis, Neisseriagonorrhoaea, Neisseria meningitidis, Orientia tsutsugamushi,Porphyromonas gingivalis, Pseudomonas aeruginosa, Staphylococcus aureus,Streptococcus agalactiae, Streptococcus pyogenes, Streptococcuspneumoniae, and Yersinia enterocolitica, vaccinia viruses, e.g., cowpoxvirus, rabbitpox virus, myxoma virus, and Shope fibroma virus, HIV-1,HPV, HTLV1, hepatitis C virus, hepatitis B virus, adeno-associatedviruses (AAV), adenoviruses, coronaviruses, coxsackieviruses,cytomegaloviruses, herpesviruses, e.g., Murid Herpesvirus-4 (MuHv-4),Kaposi's sarcoma-associated herpesvirus (KSHV), Ebstein-Barr virus(EBV), FMDV, herpes simplex viruses, e.g., HSV-1 and HSV-2, pseudorabiesvirus (PrV), pseudorabies virus, respiratory syncytial virus,rhinoviruses, sidbis viruses, flaviviruses, e.g., dengue viruses (dengue1, 3, 4), japanese encephalitis virus, kunjin virus, Murray Valleyencephalitis virus, powassan virus, St. Louis encephalitis virus,ti-borne encephalitis virus, West Nile virus, yellow fever virus,pestiviruses, e.g., viruses that cause border disease, bovine viraldiarrhea, classical swine fever, hemorrhagic fever viruses, e.g., Ebolavirus, Marburg virus, Lassa fever virus, Rift Valley virus, otherarenaviridaie viruses, other bunyaviridae viruses and other filoviridaeviruses, and parasites, e.g., Giardia lamblia, Leishmania ssp.,Encephalitozoon spp., Neospora caninum, Plasmodium ssp., Toxoplasmagondii, and Trypanosoma cruzi.

In certain aspects, the method for detecting an infectious pathogenpresent in a biological sample obtained from a subject who is suspectedof being infected, includes: (a) contacting the biological sampleobtained from the subject with an adsorption media under conditions toform an adhering complex comprising the adsorption media and thepathogen; (b) separating the adhering complex from components of thebiological sample that are not included in the complex while maintainingthe complex; and (c) collecting the pathogen of the adhering complex,wherein the pathogen or a component of the pathogen is collected afterusing an enzymatic digestion while the pathogen is resident on theadsorption media.

In some embodiments, the digestion is with one or more enzymes such as aprotease or DNase while the pathogen (e.g. virus) is still adsorbed tothe media.

H. Concentrators and Kits

In certain embodiments, the present invention provides a concentrator ina form that resembles a hypodermic syringe. The FIGURE herein is oneembodiment of the concentrator of the present invention. Theconcentrator 100 has a barrel 118 filled with adsorption media 105. Incertain instances, the barrel is a hollow cylindrical barrel portion 118with adsorbate contained therein 105, with one end terminating in anoutlet tube 117 of a diameter smaller than the diameter of thecylindrical barrel portion 118. In some instances, a hollow hypodermicneedle 112 communicates with the outlet tube 117 of the barrel portion118. In some instances, one or more different polysaccharide adsorbents,e.g., 1, 2, 3, 4, 5 or more different polysaccharide adsorbents, areattached to the adsorption media. The concentrator 100 has a plunger 130with a shaft 115 that allows the user to remove a sample from a subjectusing a needle 112 which is attached to the concentrator 100. The needlecan be of various gauges as are typical for the sample being removedfrom a subject. In certain aspects, the concentrator 100 has an optionalreservoir 110 filled with fluid such as saline, water or a buffer. Inoperation, the needle is inserted into a subject to remove a sample suchas blood. As the plunger 130 is withdrawn from the barrel 118 usingshaft 115, a breakable barrier 108 between the reservoir 110 and media105 is broken by action of the withdrawn plunger, allowing the fluid tobe drawn through the media to wet or prime the media. The fluid (e.g.,saline) is drawn through the media 105 and ends in area 121. In certaininstances, the optional reservoir 110 comprises a breakable seal orbarrier 108 such as foil, an adhesive, or plastic, disposed between theoutlet tube and adsorbate 105.

As a sample (e.g., blood) is drawn through the media, the analyte orpathogen contained therein adheres to the media. In certain instances,the withdrawn plunger 115 can be pushed by the user back toward thebarrel of the concentrator. In certain instances, the sample such as theremoved blood can be injected back and returned to the subject. Thisback and forth operation of the plunger can be repeated several times inorder to concentrate the analyte/pathogen onto the media. The operationof removing the plunger back and forth can be repeated 1, 2, 3, 4, 5, 6or even more times.

The analyte/pathogen can be removed from the media using the methodstaught herein. For example, in certain instances, normal saline can beused to remove any adhering cells and debris. Thereafter, 2N saline canbe used to remove the analyte or pathogen. In certain other instances, abuffer or saline is used to remove all adhering sample components.

In still other aspects, a sterilizing filter is used which can beattached to the outside of the concentrator 100. For example, the needlecan be removed and the filter is attached, or the needle has a filteradapter. The filter allows the concentrator to be adaptable for fielduse. For example, in the field, sterile water or fluid may not beavailable. In certain instances, impure water or fluid is drawn throughthe filter and, now sterilized through the media 105. In certaininstances, reservoir 110 is optionally removed and the sterilized wateror sterilized fluid drawn through the sterilizing filter primes or wetsthe media.

In one embodiment, the present invention provides a kit which comprisesa concentrator. In certain aspects, the kit includes a package that canbe used to hold the concentrator before and after use. The concentratorcan be used as discussed above and thereafter sent out for furtheranalysis. The kit optionally includes instructions for use.

In one aspect, the concentrator comprises:

a) a hollow cylindrical barrel portion 118 with adsorbate containedtherein 105, one end terminating in an outlet tube 117 of a diametersmaller than the diameter of the cylindrical barrel portion 118;

b) a hollow hypodermic needle 112 communicating with the outlet tube 117of said barrel portion 118;

c) a plunger 130 comprising an elastomeric material disposed within andadapted for reciprocal movement within said cylindrical barrel 118;

d) shaft means 115 attached to said plunger 130 and extending outwardlyof the end of said barrel portion 118 opposite the outlet tube 117 andoperative to impart reciprocating movement to said plunger 130 towardand away from the outlet tube of said barrel portion 118; and

f) an optional reservoir 110 with a breakable seal 108, disposed betweenthe outlet tube and adsorbate.

III. EXAMPLES

The following examples are offered to illustrate, but not to limit, theclaimed invention.

Example 1 Diagnosing Malaria in Individuals Using a Heparin ConjugatedAdsorption Media

This example illustrates the use of a heparin conjugated adsorptionmedia to detect parasitized red blood cells (pRBCs) in a blood samplefrom an individual suspected of having a malaria infection.

A whole blood sample is taken from an individual by standard methods andfollowing clinical guidelines. The blood sample (test sample) iscontacted with heparin immobilized on the surface of the adsorptionmedia under conditions to allow any parasitized red blood cells in thetest sample to attach to the immobilized heparin. Next, the unboundsample is removed without disturbing the adhering complex formed by thepRBCs and the heparin on the test adsorption media. The test media isthen observed using the naked eye to detect any change in color comparedto an adsorption media to whole blood free of parasitized red bloodcells. If a color change is detected (e.g., the test media appears morered), this indicates that the individual is likely to have a malariainfection.

The color of the test media is compared to the color of an adsorptionmedia exposed to a blood sample from a healthy control, such as anindividual that does not have a malaria infection. The individual isdiagnosed as not having a malaria infection if the test adsorption mediaand the healthy control media have a similar color. The color of thetest media is compared to the color of an adsorption media exposed to ablood sample from a positive control, such as a patient withuncomplicated malaria or severe malaria. The individual is diagnosed ashaving a malaria infection if the test adsorption media and the positivecontrol media have a similar color.

Example 2 A System for Diagnosing a Viral Infection and ExtracorporeallyTreating the Infection

This example illustrates a system of the present invention.

The system comprises at least two cartridges, wherein the firstcartridge is a smaller diagnosis cartridge and the second cartridge is alarger therapeutic cartridge. The first or diagnostic cartridge containsa short column between 3-6 ml, which is filled with heparinized beads.The first cartridge is used for the detection of a blood samplesuspected of containing hepatitis C virus (HCV).

A 2 ml blood sample is obtained from the subject. The blood sample isloaded onto the diagnosis column and forms an adhering complexcomprising the adsorption media and HCV. Thereafter, the column iswashed and with a 0.01 N saline wash buffer. The column is thereafterwashed by applying a 2N saline elution buffer to the concentrate theinfectious pathogen.

HCV RNA is detected using RT-PCR using a standard kit, such as COBAS®AmpliPrep/COBAS® TaqMan® HCV test (Roche Diagnostics, Indianapolis,Ind.) and RealTime HCV Genotype II (Abbott Molecular, Abbott Park,Ill.).

The second larger therapeutic cartridge is built with heparinized coatedbeads. A 300 ml adsorption column is fixed to a vertical stand. The 300ml of beads are then added to the cartridge and sealed.

The patient uses extracorporeal removal of the HCV using the therapeuticcolumn. Binding of hepatitis C virus to the heparin occurs. The heparinis effective to identify and remove the infectious agent.

Example 3 Processing a Sample from an Individual Suspected of BeingInfected with Ebola Virus Prior to Detecting the Virus

This example illustrates the use of an exemplary embodiment of thepresent invention for isolating Ebola virus particles from a biologicalsample from a subject infected with Ebola virus.

An Ebola infected whole blood sample is circulated over a column filledwith heparinized beads. At various time points, the flow-through fromthe column is collected and tested for the presence of Ebola. Theheparinized beads are gently washed with a 0.01N saline solution withoutdisturbing the bead matrix. The flow-through from the washing step iscollected and tested for Ebola. The flow-through contains cells such asblood cells that can interfere with the sensitivity of commercial viraldetection assays. An elution buffer containing 2N saline solution isapplied to the column and the eluent is collected. The eluent is testedfor the presence of Ebola using the CDC's protocol “Ebola Virus VP40Real-Time RT-PCR assay. This method uses a TaqMan® assay to detect theviral protein 40 of the Ebola virus.

Example 4 Processing and Concentrating Pathogens from a Blood SampleUsing Polysaccharide Adsorbent Modified Beads

This example illustrates the use of an exemplary embodiment of thepresent invention for removing and concentrating 14 different pathogensfrom a biological sample.

Approximately 0.6 grams of adsorption media, such as either heparinmedia alone, mannose media alone, polyethylenimine PEI media, acomposite of heparin and mannose media, or a composite of heparin andmannose media and naked beads was packed into 2.5 ml filter syringes(Mobicol) with 100 μm endplates. 2 ml of the pathogen suspensions inblood was prepared by culturing the pathogens (e.g., P. aeruginosa,MRSA, ESBL-producing K. pneumonia, carbapenem-resistant K pneumonia, K.pneumonia, carbapenem-resistant E. coli, E. coli, S. pneumonia, E.faecalis, vancomycin-resistant E. faecalis, E. faecium, A. baumannii, C.albicans, and CMV) overnight and diluting to the appropriateconcentrations (Table 1). The packed filter syringes were rinsed with 3ml of PBS, followed by passing the pathogen suspension over the syringesthree times. 1.0 ml of the elution buffer (2N saline solution) waspassed over the syringes and the flow-through (eluent) was collected.Standard dilution and plating techniques were used to enumerate thepathogens of the eluent. The test was replicated either two times orthree times for each media and pathogen combination that was analyzed.The results are shown in Tables 1 and 2.

TABLE 1 Concentration of Pathogen After Passage Over Modified AdsorptionMedia Pathogen Count (CFU/ml or PFU/ml) Ending Concentration StartingHep/Man Hep/Man/PEI Bacteria Concentration Heparin Mannose PEI (0.6 g)(0.6 g) P. aeruginosa 4.02E+05 5.27E+05 3.79E+05 4.73E+05 4.67E+052.75E+05 MRSA 1.21E+05 1.02E+04 1.22E+05 1.48E+04 2.03E+04 3.07E+03 K.pneumoniae 2.82E+05 1.72E+05 1.98E+05 1.80E+05 (ESBL) K. pneumoniae1.40E+05 7.83E+01 1.70E+02 2.07E+02 2.07E+02 1.88E+02 (CRE) K.pneumoniae 4.02E+05 2.55E+05 2.66E+05 1.64E+05 E. coli (CRE) 2.57E+051.90E+02 2.25E+02 1.83E+02 2.00E+02 2.27E+02 E. coli 6.15E+05 1.54E+038.92E+02 1.01E+03 S. pneumoniae 9.80E+04 4.60E+04 5.80E+04 7.48E+04 E.faecalis 6.43E+05 6.17E+03 5.83E+03 3.34E+03 E. faecalis(VRE) 6.17E+055.40E+04 5.42E+04 4.80E+04 E. faecium 9.17E+05 4.00E+05 5.67E+055.33E+05 A. baumannii 1.83E+05 3.82E+04 1.82E+04 8.50E+04 C. albicans3.35E+05 2.15E+05 CMV 7.59E+04 1.35E+04

TABLE 2 Amount of Pathogen Removed From a Blood Sample CFU or PFUremoved per gram of media Heparin Mannose PEI Hep/Man Hep/Man/PEI P.aeruginosa 0.00E+00 7.67E+04 0.00E+00 0.00E+00 4.23E+05 MRSA 3.69E+05−3.33E+03  3.54E+05 3.36E+05 3.93E+05 K. pneumoniae (ESBL) 3.67E+052.80E+05 3.40E+05 K. pneumoniae (CRE) 4.66E+05 4.66E+05 4.66E+054.66E+05 4.66E+05 K. pneumoniae 4.90E+05 4.53E+05 7.93E+05 E. coli (CRE)8.56E+05 8.56E+05 8.56E+05 8.56E+05 8.56E+05 E. coli 2.04E+06 2.05E+062.05E+06 S. pneumoniae 1.73E+05 1.33E+05 7.73E+04 E. faecalis 2.12E+062.12E+06 2.13E+06 E. faecalis (VRE) 1.88E+06 1.88E+06 1.90E+06 E.faecium 1.72E+06 1.17E+06 1.28E+06 A. baumannii 4.83E+05 5.49E+053.27E+05 C. albicans 4.00E+05 1.12E+06 CMV 2.08E+05

The tables above illustrate the removal and concentration of 14different pathogens using methods disclosed herein. For example, as isshown in Table 1, a MRSA sample has a starting concentration of 1.21×10⁵CFU/ml. Table 1 shows various efficiencies of the adsorbates of thepresent invention. After a pass or repeated passes over a column havingheparin, mannose, naked bead (PEI), a mixture of heparin/mannose, orheparin/mannose and PEI, the ending concentrations of each sample wasmeasured, recorded and tabulated.

The samples were repeatedly passed over the columns in an iterativefashion. The samples can be passed multiple times each time reducingbacteria concentrations in the sample and concentrating the bacteria orpathogen on the media.

A significant amount of pathogen is separated from the sample using theadsorption media. Table 2 is a summary table reporting the removal ofpathogens using various media. After the pathogen is concentrated on themedia column, it can be freed from the column and the its presence andidentity can be determined. The pathogen can be concentrated by applyinga volume of elution buffer that is less than the starting sample volume.Furthermore, the method can be used to remove cells and debris from theblood sample that can interfere with standard pathogen detectionmethods, such as colorimetric assays, immunoassays, ELISAs, PCR-basedassays, and the like.

Example 5 Identification of a Pathogen Such as a Virus in a BiologicalSample Using Polysaccharide Adsorbent Modified Beads

This example illustrates the use of an exemplary embodiment of thepresent invention for removing a pathogen such as a virus (e.g.,hepatitis C virus (HCV)) from a biological sample.

In this example, a digestion step is employed with one or more enzymessuch as a protease or DNase while the pathogen (e.g. virus) is stilladsorbed to the media. In this manner, the digestion step is performedwith a much smaller volume of buffer solution i.e., much smaller thanrequired compared to solutions needed for elution. The protease can befor example, chymotrypsin or trypsin; the DNase can be an exo orendonuclease or a restriction enzyme.

In this example, a pathogen such as a virus is concentrated on theadsorbent beads. The virus is digested with one or more enzymes whileadsorbed directly on the beads. The beads are removed and PCR isperformed. The steps are as follows:

-   -   1) Infected whole blood, serum, plasma, or other biological        fluid is passed over heparinized beads (or other affinity        ligand);    -   2) The biological fluid can be exposed to the heparinized beads        either by passage through a small device containing the beads        (e.g., the FIGURE herein), or alternatively, the adsorbent beads        are added to a vessel containing a biological fluid (e.g. a well        in a multi-plate reader, conical vial, test tube, etc.);    -   3) The virus binds to heparin (or other carbohydrate or affinity        ligand);    -   4) The whole blood, serum, plasma, or other biological fluid is        then removed from the beads by rinsing with normal saline;        -   a. For a small device containing the beads, saline is passed            over the beads using flow.        -   b. For beads floating in a biological fluid, the beads can            be separated or filtered and added to a secondary container            for washing.    -   5) The beads with bound pathogens are then exposed to enzyme(s)        for digestion;    -   6) The beads are removed and a PCR amplification step is        performed for analysis.

It is also possible to apply this approach to a test strip with atextured heparinized (or other ligand) on the surface of the test strip.

Pathogens such as bacterial, viral, fungal, protozoan, parasitic, andmicrobial pathogens can be detected using assays, such as, colorimetricassays, immunoassays (e.g., sandwich assays or dipstick assay),enzyme-linked immunosorbent assays (ELISAs), PCR-based assays (e.g.,RT-PCR, qPCR, TaqMan® assays), pathogen growth assays (e.g., drugresistance or antibiotic resistance assays), and variants thereof.

HCV RNA is detected using RT-PCR using a standard kit, such as COBAS®AmpliPrep/COBAS® TaqMan® HCV test (Roche Diagnostics, Indianapolis,Ind.) and RealTime HCV Genotype II (Abbott Molecular, Abbott Park,Ill.).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference.

1. An in vitro method for concentrating infectious pathogens present ina biological sample obtained from a subject who is suspected of beinginfected with said pathogens, the method comprising: (a) contacting thebiological sample obtained from the subject with an adsorption mediaunder conditions to form an adhering complex comprising the adsorptionmedia and said pathogens; (b) separating the adhering complex fromcomponents of the sample that are not included in the complex whilemaintaining the complex; (c) washing the adhering complex with a washbuffer; and (d) collecting pathogens of the adhering complex by applyingan elution buffer to the complex, thereby concentrating the infectiouspathogens in an eluent.
 2. The method of claim 1, wherein the washbuffer is a saline solution.
 3. The method of claim 1, wherein theelution buffer is a high ionic strength or hypertonic saline solution.4. The method of claim 1, wherein the biological sample is selected fromthe group consisting of whole blood, serum, plasma, urine, feces,sputum, tears, saliva, bronchial lavage fluid, other bodily fluid, andcombinations thereof.
 5. The method of claim 1, wherein the adsorptionmedia is a solid substrate of high surface area having at least onepolysaccharide adsorbent on the surface thereof.
 6. The method of claim5, wherein the at least one polysaccharide adsorbent is attached to thesurface of the solid substrate by end-point attachment.
 7. The method ofclaim 5, wherein said solid substrate comprises a plurality of rigidpolymer beads.
 8. The method of claim 7, wherein said plurality of rigidpolymer beads are rigid polyethylene beads.
 9. The method of claim 6,wherein the at least one polysaccharide adsorbent is a member selectedfrom the group consisting of heparin, heparan sulfate, mannose, dextransulfate, hyaluronic acid, salicylic acid, chitosan, and a combinationthereof.
 10. The method of claim 9, wherein the mannose is D-mannose ora D-mannose polymer.
 11. The method of claim 9, wherein the at least onepolysaccharide adsorbent is heparin and mannose.
 12. The method of claim1, wherein said pathogens are selected from the group consisting ofPlasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodiummalariae, Ebola virus (EBOV), filovirus, Flaviviridae, Streptococcusaureus, Escherichia coli, carbapenem-resistant enterobacteriaceae (CRE)bacteria, an ESBL-producing pathogen, vancomycin-resistant enterococci(VRE) bacteria, Acinetobacter baumannii, Klebsiella pneumoniae,Klebsiella oxytoca, Enterococcus faecalis, Enterococcus faecium, Candidaalbicans, cytomegalovirus (CMV), Adenovirus, herpes simplex virus 1(HSV1), herpes simplex virus 2 (HSV2), and any combination thereof. 13.The method of claim 1, further comprising detecting the isolatedinfectious pathogens.
 14. The method of claim 13, wherein detecting theisolated infectious pathogens comprises a colorimetric assay, animmunoassay, an enzyme-linked immunosorbent assay (ELISA), a PCR-basedassay, a pathogen growth assay, or a combination thereof.
 15. Aconcentrator for use according to the method of claim 1 comprising abarrel filled with adsorption media, a plunger and a needle.
 16. A kitcomprising the concentrator of claim 15, a wash buffer and an elutionbuffer.
 17. An in vitro method for reducing blood cells from abiological sample obtained from a subject who is suspected of beinginfected with an infectious pathogen, the method comprising: (a)contacting the biological sample obtained from the subject with anadsorption media under conditions to form an adhering complex comprisingthe adsorption media and a pathogen present in the sample; and (b)separating the blood cells of the sample and the adhering complex whilemaintaining the adhering complex, thereby reducing blood cells of thesample. 18-31. (canceled)
 32. An in vitro method for diagnosing malariain a subject who is suspected of being infected with Plasmodium, themethod comprising: (a) contacting a sample obtained from said subjectwith an adsorption media under conditions to form an adhering complexcomprising the adsorption media and a cell present in the sample whichis infected with Plasmodium; (b) determining the presence of theadhering complex by detecting a physical change to the adsorption media;and (c) predicting that the subject has malaria based on the physicalchange to the adsorption media compared to a reference adsorption mediathat has been contacted with a control sample. 33-44. (canceled)
 45. Anin vitro method for determining that a subject is infected with aninfectious pathogen, the method comprising: (a) contacting a whole bloodsample obtained from said subject with an adsorption media to form anadhering complex comprising the adsorption media and a pathogen presentin the sample; and (b) determining presence of the adhering complex bydetecting a physical change to the adsorption media; and (c) predictingthat the subject is infected by the infectious pathogen based on thephysical change to the adsorption media compared to a referenceadsorption media that has been contacted with a control sample. 46-55.(canceled)
 56. A kit, the kit comprising: a concentrator; and packagingfor before and after use.
 57. (canceled)
 58. An in vitro method fordetecting an infectious pathogen present in a biological sample obtainedfrom a subject who is suspected of being infected with said pathogen,the method comprising: (a) contacting the biological sample obtainedfrom the subject with an adsorption media under conditions to form anadhering complex comprising the adsorption media and said pathogen; (b)separating the adhering complex from components of the biological samplethat are not included in the complex while maintaining the complex; and(c) collecting the pathogen of the adhering complex, wherein thepathogen or a component of the pathogen is collected after using anenzymatic digestion while the pathogen is resident on the adsorptionmedia. 59-74. (canceled)